Phusion polymerase

The final concentration of each primer in a reaction using Phusion DNA Polymerase may
be 0.2–1 μM, while 0.5 μM is recommended.

During thermocycling, the denaturation step should be kept to a minimum. Typically, a 5–10 second denaturation at 98°C is recommended for
most templates.

Annealing:
Annealing temperatures required for use with Phusion tend to be higher than with other PCR polymerases. The NEB Tm calculator should
be used to determine the annealing temperature when using Phusion. Typically, primers greater than 20 nucleotides in length anneal
for 10–30 seconds at 3°C above the Tm of the lower Tm primer. If the primer length is less than 20 nucleotides, an annealing temperature
equivalent to the Tm of the lower primer should be used. A temperature gradient can also be used to optimize

Insert-specific primers only: For verifying presence of insert directly. Should only give amplicon if insert is present. However, if the insert was tranferred from one vector to another, colony PCR will give a positive result (amplicon) even if the vector is wrong

Backbone-specific primers: Altough the presence of the insert can't be confirmed directly in this way, it may still be useful to do colony PCR with primers specific to the regions in the vector flanking the insert, checking that the new vector gives the expected amplicon size.

Combination of insert- and backbone-specific primers: Useful in the case of short insert sequences. The presence of the insert can be verified directly while giving a longer, selectable amplicon size. Use the parent vector as a negative control template.

Emulsion PCR

RT-PCR

qPCR

Digital PCR

Inverse PCR

PCR program design

Advantages of lower annealing temperature:

Possible higher yield

Shorter run time

Disadvantages of lower annealing temperatures:

Higher danger of mis-priming and primer dimers

Longer run time.

For short amplicons, a two-step cycling program may be tried. Given denaturation at 95 C and annealing at 50-55 C, the polymerase may have time to produce a product during the ramping phase from annealing to denaturation.

To do a quick, rough check on the thermocycling performance and corresponence between displayed and actual temperature, run a PCR program with a hold at 4 C at the end. Immediately after the display shows that 4C is reached and that the machine is holding the temperature, open the cover and feel the temperature of the heatblock. If it isn't cold, there is a significant time-lag/discorrespondence between the displayed and heatblock surface temperatures.

Purification of oligomers

Reverse-phase cartridge purification (Sigma:"RP1"). Separates truncated and full-length products on the basis of difference in hydrophobicity between full-length products with DMT protecting group present, and truncated sequences without DMT group. Unsuited for longer oligomers, as the proportion of DMT-containing truncated sequences increase with oligomer length. From Sigma website:

"As the oligo length increases, the proportion of uncapped products (truncated sequences bearing the DMT) tends to increase. These impurities will not be removed by RP1 and thus for longer oligos, HPLC or PAGE is recommended."

HPLC reverse-phase:

From Sigma website:

"The resolution based on lipophilicity will decrease with the length of the oligo. Therefore, RP-HPLC is usually not recommended for purifying products longer than 50 bases. Although longer oligos (up to 80 bases) can be purified using this method, the purity and yields may be adversely affected."

PAGE:

From Sigma website:

"This technique is recommended when a highly purified product is required. PAGE is the recommended purification for longer oligos (≥50 bases)."

Anion-Exchange HPLC:

From Sigma website:

"Anion- Exchange HPLC is limited by length (usually up to 40mers). The longer the oligonucleotide the lower the resolution on the Anion-Exchange HPLC column and thus the purity of the target oligo."

Comparison of calculated and reported Tm values

Oligomer

Sequence

Tm (C) [calculated]

Tm (C) [Analytical]

Supplier

GFP-END-FWD

65.6(1)

65.2

Sigma

GFP-END-REV

59.4(1)

58.9

Sigma

GFP-END-LVA-REV

84.5(1)

84.5

Sigma

pSB-SeqA

TGCAAGAAGCGGATACAG

60.7

60.2

Sigma

LacUV5_49bp_R_FWD

caaccggtGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCG

88.3(1)

88.3

Sigma

LacUV5_49bp_R_REV

gtacatgtTCCACACATTATACGAGCCGGAAGCATAAAGTGTA

78.2(1)

78.1

Sigma

ArgI46bp_R_FWD

caaccggtGCTTTAGACTTGCAAATGAATAATCATCCATAT

77.2(1)

77.2

Sigma

ArgI46bp_R_REV

gtacatgtTAAAATTCAATTTATATGGATGATTATTCATTT

67.8(1)

67.6

Sigma

rrnB p1_74bp_FWD

agccgggcgatgccaaccggGTTGCGCGGTCAGAAAATTA

91.2(1)

91.3

Sigma

rrnB p1_74_bp_REV

ctccattattattgtacatgAGTGGTGGCGCATTATAGG

75.7(1)

75.6

Sigma

GreA_60bp_FWD

agccgggcgatgccaaccggGGCGCAACGCCCTATAAAGT

91.5

91.6

Sigma

GreA_60bp_REV

ctccattattattgtacatgATAGTCATTTTACCCTGAAGTTCCC

74.5

74.5

Sigma

LacUV5_49bp_FWD

AGCCGGGCGATGCCAACCGGgcaccccaggctttacactttatgcttccggctcg

95.0(1)

95.5

Sigma

LacUV5_49bp_REV

CTCCATTATTATTGTACATGtccacacaTTatacgagccggaagcataaagtgta

80.3(1)

80.3

Sigma

pJP-1 seq5

CAGCGTGCGAGTGATTAT

60.6(1)

53.9

Macrogen

pJP-1 seq6

AGACCACATGGTCCTTCT

57.5(1)

53.9

Macrogen

COPCR1FWD2

TAATCGCCTTGCAGCACATC

55.5(1)

58.4

Macrogen

COPCR1REV

TTGCATCACCTTCACCCTCT

65.1(1)

58.4

Macrogen

SeqMG1

AGCAGATCCACATCCTTGAA

62.7(1)

56.4

Macrogen

rrnB_p1_long_FWD

agccgggcgatgccaaccggGTATCCTACGCCCGTGGTTA

90.6(1)

85.1

Macrogen

GreA_long_FWD

agccgggcgatgccaaccggTCACCCTTAAGTACGCCGTT

89.5(1)

84.0

Macrogen

RF-LVA-EcoRI-FWD

GGGATTACACATGGCATGGATGAACTATACAAAGCAGCAAACGACGAAAACT

84.0(1)

80.5

Macrogen

Observations: For deliveries from Sigma Aldrich, Tm values from the Finnzymes Tm calculator and reported by Sigma are in good agreement. For deliveries from Macrogen, the reported Tm values systematically lower than the values calculated by Finnzymes Tm calculator - up to 6 degrees lower, and an average of 4,4 degrees lower. Does the synthesis process at Macrogen (impurities?) cause lower Tm values?